Targeting of endoplasmic reticulum dysfunction and protein folding stress to treat neurological conditions

ABSTRACT

Methods and therapeutic compositions are disclosed for treating neurological disorders, such as Amyotrophic Lateral Sclerosis (ALS), Alzheimer&#39;s disease, Parkinson&#39;s disease and/or Huntington&#39;s disease, using Salubrinal analogs, or pharmaceutically acceptable salts, hydrates, or solvates thereof.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/US2019/018161, filed on Feb. 15, 2019,which claims priority to U.S. Provisional Patent Application Ser. No.62/646,112 filed Mar. 21, 2018. The contents of the aforementionedapplications are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

The technical field of this invention is the treatment ofneurodegenerative or neuromuscular disorders such as Amyotrophic LateralSclerosis (ALS), Alzheimer's disease, Parkinson's disease and/orHuntington's disease.

ALS is a progressive neurological disorder characterized by muscle fiberatrophy resulting from the degeneration of motor neurons in the spinalcolumn and brain. ALS affects approximately 30,000 US citizens, withonly about 10% of cases being classified as the familial form of ALS. Ina subset of familial patients with mutations in the metabolic enzymesuperoxide dismutase 1 (SOD1), the pathological progression may beattributed to unknown factors associated with a mutant form of the SOD1enzyme. However, in the majority of ALS cases the SOD1 gene contains nomutations and the mechanism of disease pathology is even less clear.Therefore, the remaining 90% of ALS cases are classified as sporadiccases, with no well-characterized genetic component or causal agent.

Alzheimer's disease is another progressive neurodegenerative disease.Early symptoms include difficulty in remembering recent events and inmany instances Alzheimer's disease progresses to dementia. The diseaseprocess is associated with plaque deposits of the β-amyloid peptide andneurofibrillary tangles of the microtubule binding protein tau in thebrain.

Amyloid protein accumulations have also been implicated in Parkinson'sand Huntington's diseases. In Parkinson's disease, misfolding of theα-synuclein protein has been associated with disease manifestation. InHuntington's disease, alterations in Huntingtin protein appear to play arole in disruption of nerve cell functions.

Recently, it has been suggested that endoplasmic reticulum (ER) stressand the unfolded protein response (UPR) are involved in these and otherneurological diseases. The endoplasmic reticulum is a membrane systemwithin the cytoplasm of eukaryotic cells and serves various functions,including the synthesis, folding, modification, and transport ofproteins. A number of stress conditions can interfere with ER functionand cause abnormal protein folding, resulting in a cellular conditiontermed “ER stress.” The unfolded protein response (UPR) is related to ERstress and is generally understood to be triggered by an accumulation ofunfolded or misfolded proteins within the cell.

Studies have identified several signaling branches of UPR, including,for example, increased phosphorylation of eukaryotic initiation factor2-alpha (eIF2-alpha). Eukaryotic translation initiation factor 2-alphakinase 3, also known as protein kinase R (PKR)-like endoplasmicreticulum kinase (PERK), is an enzyme that phosphorylates eIF2-alpha.Over phosphorylation of eIF2-alpha has also been demonstrated inpost-mortem spinal cord of ALS patients. Additionally, increasedexpression of X-box binding protein 1 (XBP1), activated transcriptionfactor 4 (ATF4), and transcription factor C/EBP homologous protein(CHOP) expression is often observed in human ALS spinal cord analysesand these factors may also be implicated in ER stress and UPR.

Activation of the UPR can also be modeled in transgenic mice that overexpress mutant human SOD1 genes. Genetic modulation of the UPR intransgenic mice over-expressing mutant human SOD1 significantlyinfluences motor neuron disease symptom onset and lifespan. Miceover-expressing G85R mutant human SOD1 in the context of PERKhaplo-insufficiency have earlier disease onset and reduced lifespanscompared to G85R-SOD1 mice on a PERK+/+ background. On the other hand,the same G85R-SOD1 mice crossed to mice with a mutated GADD34 gene onone allele have delayed onset and significantly prolonged survival.

Salubrinal(N-(2,2,2-trichloro-1-(3-(quinolin-8-yl)thioureido)ethyl)cinnamamide) isa small molecule identified as protective of ER stress in a cell basedassay from a library of 19,000 compounds See, Boyce et al., A selectiveinhibitor of eIF2alpha dephosphorylation protects cells from ER stress,Science, 307:935-939, (2005). Specifically, Salubrinal has been shown toinhibit ER stress-mediated apoptosis that is induced by tunicamycin, aprotein glycosylation inhibitor.

At least one study suggests that salubrinal, as an inhibitor of ERstress, slowed motor neuron degeneration and extended survival of SOD1mice. See, Walker et al. PloS One 8 (11) e81170 (2013).

Salubrinal has also been reported to attenuate β-amyloid-inducedneuronal death and microglial activation associated with Alzheimer'sdisease. See, Huang et al., Neurobiol. Aging, May 33(5) 1007 (2012).Salubrinal has further been reported to protected PC12 cells from celldeath induced by α-synuclein, a component of Lewy bodies seen inParkinson's disease. See, Smith et al., Human Molecular Genetics 14(24)3801-11 (2005).

The chemical structure of Salubrinal is shown below in Formula (I):

Despite its use as a research tool, use of Salubrinal as a therapeuticagent is limited because of its low solubility in aqueous solutions,rapid clearance and relatively high toxicity in animals (EC50˜15 μM).New chemical compounds with better properties such as, for example andwithout limitation, higher solubility in biological fluids, slowerclearance rates, and lower toxicity are required.

SUMMARY OF THE INVENTION

Methods and compositions are disclosed that relate to the treatment ofneurological (neurodegenerative or neuromuscular) disorderscharacterized by endoplasmic reticulum stress or unfolded proteinresponse.

In one aspect, methods and therapeutic compositions for treatingneurological disorders by administering a compound are disclosed.Preferred compounds belong to a class of Salubrinal analogs (excludingSalubrinal itself) and can be defined by the general Formula (II):

In certain embodiments, R¹ can be H, C₃₋₆ cycloheteroalkyl, or N(CH₃)₂,R² can be H, C₃₋₆ cycloheteroalkyl, or when combined with R⁴ is together—CH(C₁₋₁₀ alkyl)- or —CH(C₁₋₅heteroalkyl)-, R³ can be H, CH₂(C₃₋₆cycloheteroalkyl), C₃₋₆ cycloheteroalkyl, amino acid, amino acidderivative, C₁₋₂₀ alkyl, or C₁₋₂₀ heteroalkyl, R⁴ can be null, H,CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆ cycloheteroalkyl, amino acid, aminoacid derivative, C₁₋₂₀ alkyl, or C₁₋₂₀ heteroalkyl, R⁵ can be ═S or SR⁶,R⁶ can be CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆ cycloheteroalkyl, acid, aminoacid derivative, C₁₋₂₀ alkyl, or C₁₋₂₀ heteroalkyl, A-B can be CH—CH₂ orC═CH, and D-E can be C—N, CH—N, or C═N.

In one aspect of the invention the salubrinal analogs are designed todecompose in plasma over time to release salubrinal. Thus, thecompositions are novel prodrug versions of salubrinal.

In other embodiments, the compound can be a pharmaceutically acceptablesalt, hydrate, or solvate thereof. For example, the compound can be achloride salt, hydrochloride salt, or a phosphate salt.

In one exemplary embodiment, the compound can have the structure ofFormula (III), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (IV), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (V), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (VI), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (VII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (VIII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (IX), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (X), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (XI), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (XII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In another exemplary embodiment, the compound can have the structure ofFormula (XIII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Methods and therapeutic compositions are also disclosed for modulatingor ameliorating endoplasmic reticulum stress in neurodegenerative orneuromuscular disorders, and particularly Amyotrophic Lateral Sclerosis(ALS), Alzheimer's disease, Parkinson's disease and/or Huntington'sdisease. In one aspect, administration of the compound can inhibitendoplasmic reticulum stress-mediated apoptosis.

In one embodiment, this can be achieved through preventingdephosphorylation of the eukaryotic translation initiation factor 2alpha (eIF2-alpha) by administering an inhibitor of eIF2-alpha, such asthe salubrinal analogs described herein above as formula (II).

In another embodiments, the compound can be formulated with apharmaceutically acceptable diluent, adjuvant, or carrier. The compoundcan be formulated for intraperitoneal administration, intravenousadministration, anal administration, or any combination thereof. Thecompound can be formulated for oral administration, a single daily dose,and/or a dosage between 0.01 to 1 mg/kg/day or between 0.1 to 0.5mg/kg/day. The compositions can be administered as a single dose or inmultiple doses and can further include a pharmaceutically acceptablecarrier. Examples of carrier include, but are not limited to, lipid,lipid derivative, liposome, protein, albumin, synthetic and/or naturalpolymer, synthetic and/or natural oligomer, cyclodextrin, cyclodextrinderivative, cellulose, and cellulose derivatives. The carrier can beselected from the group consisting of sulfobutylether beta cyclodextrin,methylcellulose, or a combination thereof. The formulation can have fromabout 0.1% to about 10% of carrier, from about 0.1% to about 5% ofcarrier, from about 0.1% to about 1.0% of carrier, or from about 0.5% toabout 1.0% of carrier. In other embodiments, methods to protect cells oranimals from target cell damage induced by the exposure to xenotoxicantsknown to induce endoplasmic reticulum stress and the subsequent unfoldedprotein are disclosed. The xenotoxicants include but are not limited tochemicals and pollutants (e.g., cadmium, arsenic, paraquat, rotenone,epoxides, dioxins, cigarette smoke, etc.) and drugs (e.g., cisplatin,cyclosporine, etcetera).

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various embodiments is provided herein belowwith reference, by way of example, to the following drawings. Theskilled person in the art will understand that the drawings, describedbelow, are for illustration purposes only. The drawings are not intendedto limit the scope of the applicant's teachings in any way.

FIG. 1 is a diagram showing the synthesis of compound ID 1912;

FIG. 2 is a diagram showing the synthesis of compound ID 1913;

FIG. 3 is a table showing the MS/MS analysis of compound IDs 1901-1948;

FIG. 4A is a table showing the stability of compound IDs 1901-1931 inphosphate buffer and mouse plasma;

FIG. 4B is a table showing the stability of compound IDs 1932-1948 inphosphate buffer and mouse plasma;

FIG. 4C is a table showing the stability of two reference compounds(salubrinal and diltiazem) in phosphate buffer and mouse plasma;

FIG. 5A is a table showing the stability of compound IDs 1901-1931 inmouse liver microsomes;

FIG. 5B is a table showing the stability of compound IDs 1932-1948 inmouse liver microsomes;

FIG. 5C is a table showing the stability of three reference compounds(salubrinal, verapamil, and diphenhydramine) in mouse liver microsomes;

FIG. 6A is a table showing the cell viability in the absence oftunicamycin for compound ID 1934 at 0 μM, 1 μM, 10 μM, and 30 μM doses;

FIG. 6B is a drawing showing the area of cell viability change in theabsence of tunicamycin for compound ID 1934;

FIG. 7 is a drawing showing the cell viability area in the absence oftunicamycin for compound IDs 1901-1948 and salubrinal controls Sal 1-7and Salu;

FIG. 8 is a drawing showing the total cell viability area change in theabsence of tunicamycin for compound IDs 1901-1948 and salubrinalcontrols Sal 1-7 and Salu;

FIG. 9A is a table showing the cell viability in the presence oftunicamycin for compound ID 1934 at 0 μM, 1 μM, 10 μM, and 30 μM doses;

FIG. 9B is a drawing showing the area of cell viability change in thepresence of tunicamycin for compound ID 1934;

FIG. 10 is a drawing showing the cell viability area in the presence oftunicamycin for compound IDs 1901-1948 and salubrinal controls Sal 1-7and Salu;

FIG. 11 is a drawing showing the total cell viability area change in thepresence of tunicamycin for compound IDs 1901-1948 and salubrinalcontrols Sal 1-7 and Salu;

FIG. 12 is a drawing showing the dose-related change in cell viabilityarea in the absence of tunicamycin for compound ID 1949 and salubrinalcontrol Salub;

FIG. 13 is a drawing showing the total cell viability area change in theabsence of tunicamycin for compound ID 1949 and salubrinal controlSalub;

FIG. 14 is a drawing showing the dose-related change in cell viabilityarea in the presence of tunicamycin for compound ID 1949 and salubrinalcontrol Salub;

FIG. 15 is a drawing showing the total cell viability area change in thepresence of tunicamycin for compound ID 1949 and salubrinal controlSalub;

FIG. 16 is a drawing showing the cell viability at differentconcentrations of tunicamycin for compound IDs 1901, 1906, 1912, and1913 and salubrinal Salub;

FIG. 17 is a drawing showing the cell viability at differentconcentrations of tunicamycin without compound or salubrinal;

FIG. 18 is a drawing showing the cell viability in the absence oftunicamycin for compound IDs 1901, 1906, 1912, and 1913 and salubrinalSalub;

FIG. 19 is a drawing showing the cell viability at differentconcentrations of tunicamycin for compound IDs 1914, 1918, 1924, and1946 and salubrinal Salub;

FIG. 20 is a drawing showing the cell viability at differentconcentrations of tunicamycin without compound or salubrinal;

FIG. 21 is a drawing showing the cell viability in the absence oftunicamycin for compound IDs 1914, 1918, 1924, and 1946 and salubrinalSalub;

FIG. 22 is a drawing showing the cell viability at differentconcentrations of tunicamycin for compound ID 1949 and salubrinal;

FIG. 23 is a drawing showing the cell viability at differentconcentrations of tunicamycin without compound or salubrinal;

FIG. 24 is a drawing showing the cell viability in the absence oftunicamycin for compound ID 1949 and salubrinal; and

FIG. 25 is a drawing showing a summary of the properties of compound IDs1901, 1903, 1905, 1906, 1912, 1913, 1914, 1918, 1924, and 1946.

DETAILED DESCRIPTION

The following abbreviations are used throughout the specifications andknown to those skilled in the art: Amyotrophic Lateral Sclerosis (ALS);super oxide dismutase-1 (SOD1); endoplasmic reticulum (ER), unfoldedprotein response (UPR), eukaryotic initiation factor 2-alpha(eIF2-alpha), eukaryotic translation initiation factor 2-alpha kinase 3,also known as protein kinase R (PKR)-like endoplasmic reticulum kinase(PERK), X-box binding protein 1 (XBP1), activated transcription factor 4(ATF4), and transcription factor C/EBP homologous protein (CHOP).

In the description that follows, and in documents incorporated byreference, a number of terms are used extensively. The followingdefinitions are provided to facilitate understanding of the methods andcompositions disclosed herein.

As used herein, the term “subject” is a human or other animal, having aneurological disorder. In some embodiments, the subjects are mammals.Examples of subjects can include, but are not limited to, humans,horses, monkeys, dogs, cats, mice, rates, cows, pigs, goats and sheep.In some embodiments, “subjects” are generally human patients having ALS.

As used herein, the term “alkyl” includes straight chain alkyl group,branched alkyl group, cyclic alkyl group, or any combinations thereof.The alkyl group may be fully saturated, monounsaturated orpolyunsaturated, the alkyl group may be unsubstituted, monosubstitutedor polysubstituted, and the alkyl group may include divalent ormultivalent radicals having the number of carbon atoms designated (i.e.,C₃₋₆ means three to six carbons). In some embodiments, the alkyl groupis a C₁₋₂₀ alkyl group, more preferably a C₁₋₁₅ alkyl group, morepreferably still a C₁₋₁₂ alkyl group, more preferably still, a C₁₋₆alkyl group, or more preferably a C₁₋₃ alkyl group. In otherembodiments, the alkyl group is a cyclic alkyl group and has at leastthree elements. Examples of saturated alkyl groups include, but are notlimited to, groups such as methyl, ethyl, propyl, isopropyl, n-butyl,isobutyl, s-butyl, tert-butyl, homologs and isomers of, for example,n-pentyl and n-hexyl, and the like. An unsaturated alkyl group is onehaving one or more double bonds (also referred to herein as “alkenyl”group) or triple bonds (also referred to herein as “alkynyl” group).Examples of unsaturated alkyl groups include, but are not limited to,vinyl, 2-propenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. The term “alkyl,” unless otherwise noted,is also meant to include those derivatives of alkyl defined in moredetail below, such as “heteroalkyl” and “heterocycle.”

As used herein, the term “alkenyl,” by itself of in combination withanother term, means, unless otherwise stated, a group containing one ormore carbon-carbon double bonds. Preferably the alkenyl group is a C₂₋₂₀alkenyl group, more preferably a C₂₋₁₅ alkenyl group, more preferably aC₂₋₁₂ alkenyl group, more preferably a C₂₋₆ alkenyl group, or morepreferably a C₂₋₃ alkenyl group. The alkenyl group can be optionallysubstituted.

As used herein, the term “alkynyl,” by itself of in combination withanother term, means, unless otherwise stated, a group containing one ormore carbon-carbon triple bonds. Preferably the alkynyl group is a C₂₋₂₀alkynyl group, more preferably a C₂₋₁₅ alkynyl group, more preferably aC₂₋₁₂ alkynyl group, more preferably a C₂₋₆ alkynyl group, or morepreferably a C₂₋₃ alkynyl group. The alkynyl group can be optionallysubstituted.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, an alkyl group consisting of the statednumber of carbon atoms and at least one heteroatom selected from thegroup consisting of oxygen (O), nitrogen (N), silicon (Si), sulfur (S),and phosphorus (P), and wherein the O, N, S, Si, and P atoms mayoptionally be oxidized and the nitrogen heteroatom may optionally be aprimary, secondary, tertiary, quaternary amine. The heteroatom(s) O, N,S, Si, and P may be placed at any interior position of the heteroalkylor at the position at which the alkyl group is attached to the remainderof the molecule. The heteroalkyl group can be optionally substituted.

The term “aryl,” by itself or in combination with another term, means,unless otherwise stated, a monovalent aromatic hydrocarbon radical of6-20 carbon atoms derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Aryl includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic or heterocyclicring. Exemplary aryl groups include, but are not limited to, radicalsderived from benzene, naphthalene, anthracene, biphenyl, indene, indane,1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthalene, and the like. Arylgroups may be optionally substituted independently with one or moresubstituents described herein.

As used herein, the term “heteroaryl,” by itself or in combination withanother term, means, unless otherwise stated, an aryl group that containthe stated number of carbon atoms and at least one heteroatom selectedform nitrogen (N), oxygen (O), sulfur (S), and silicon (Si). Theheteroatom(s) O, N, S, and Si may be placed at any position of theheteroaryl or at the position at which the aryl group is attached to theremainder of the molecule. Examples of heteroaryl groups include, butare not limited to, pyrrole, pyrazole, pyrimidine, pyrazine, pyridine,quinoline, thiophene, 1,2,3-triazole, 1,2,4-triazole, thiazole, oxazole,iso-thiazole, iso-oxazole, imidazole, furan and the like. The heteroarylgroup can be optionally substituted.

As used herein, the term “halo” or “halogen,” by themselves or as partof another substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom.

The term “substituted” refers to a chemical group, such as alkyl,cycloalkyl aryl, and the like, wherein at least one hydrogen is replacedwith a substituent as described herein, for example, halogen, azide,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino,nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone,aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,—CF₃, —CN, or the like. The term “substituted” is also contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. Illustrative substituentsinclude, for example, those described herein above. The permissiblesubstituents may be one or more and the same or different forappropriate organic compounds. For purposes of this disclosure, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. This disclosure is not intendedto be limited in any manner by the permissible substituents of organiccompounds. In many embodiments, however, any single substituent hasfewer than the 100 total atoms. In many embodiments, however, any singlesubstituent has fewer than the 10 total atoms.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

As used herein, the neutral forms of the compounds may be regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

The term “treatment” or “treating” as used herein is intended toencompass preventing the onset, slowing the progression, reversing orotherwise ameliorating a neurological (neurodegenerative andneuromuscular) disorder. In one exemplary embodiment, the neurologicaldisorder being treated is ALS.

The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of thecomposition may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of thepharmacological agent to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the pharmacological agent are outweighed by thetherapeutically beneficial effects.

The term “VialncTot” refers to the total increase in cell viability(AUC) in the presence of tunicamycin dose-response-induced proteostasis.

The term “CmpViaTot” refers to the total change in cell viability (AUC)caused by compound alone in the absence of tunicamycin.

The term “PlossBuff” refers to the percent of parent compound lost whenincubated in phosphate buffer.

The term “SgainBuff” refers to the percent of parent compound convertedto salubrinal when incubated in phosphate buffer.

The term “PlossPlas” refers to the percent of parent compound lost whenincubated in mouse plasma.

The term “SgainPlas” refers to percent of parent compound converted tosalubrinal when incubated in mouse plasma.

The term “Liver-” refers to the percent of parent compound lost whenincubated in mouse liver microsomes without NADPH cofactor.

The term “Liver+” refers to the percent of parent compound lost whenincubated in mouse liver microsomes with NADPH cofactor present.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined—e.g., the limitations of the measurement system, or thedegree of precision required for a particular purpose. For example,“about” can mean within 1 or more than 1 standard deviations, as per thepractice in the art. Alternatively, “about” can mean a range of up to20%, preferably up to 10%, more preferably up to 5%, and more preferablystill up to 1% of a given value. Where particular values are describedin the application and claims, unless otherwise stated, the term “about”meaning within an acceptable error range for the particular value shouldbe assumed.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the,” include plural referents unless the context clearly indicatesotherwise. Thus, for example, reference to “a molecule” includes one ormore of such molecules and reference to “the method” includes referenceto equivalent steps and methods known to those of ordinary skill in theart that could be modified or substituted for the methods describedherein.

Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition inwhich patients progressively lose all motor function. Evidence isaccumulating that as a result of the aging process the body increasinglyloses the ability to adequately degrade mutated or misfolded proteins.The proteasome is the piece of biological machinery responsible for mostnormal degradation of proteins inside cells. Age related loss offunction or change of function of the proteasome is now thought to be atthe heart of many neurodegenerative conditions, including Alzheimer'sdisease, Parkinson's disease, Huntington's disease, and ALS.

ALS, also called Lou Gehrig's disease, affects the motor neurons of thecortex, brain stem and spinal cord. (Hirano, A., “Neuropathology of ALS:an overview,” Neurology, 47(4 Suppl. 2): S63-6 (1996)). ALS affectsapproximately 30,000 Americans with nearly 8,000 deaths reported in theUS each year. ALS remains one of the most devastating diseases andadvances in treatment are desperately needed.

The cardinal feature of ALS is the loss of spinal motor neurons, whichcauses the muscles under their control to weaken and waste away leadingto paralysis. ALS has both familial (5-10%) and sporadic forms and thefamilial forms have now been linked to several distinct genetic loci(Deng, H. X., et al., “Two novel SOD1 mutations in patients withfamilial amyotrophic lateral sclerosis,” Hum. Mol. Genet., 4(6): 1113-16(1995); Siddique, T. and A. Hentati, “Familial amyotrophic lateralsclerosis,” Clin. Neurosci., 3(6): 338-47(1995); Siddique, T., et al.,“Familial amyotrophic lateral sclerosis,” J. Neural Transm. Suppl., 49:219-33(1997); Ben Hamida, et al., “Hereditary motor system diseases(chronic juvenile amyotrophic lateral sclerosis). Conditions combining abilateral pyramidal syndrome with limb and bulbar amyotrophy,” Brain,113(2): 347-63 (1990); Yang, Y., et al., “The gene encoding alsin, aprotein with three guanine-nucleotide exchange factor domains, ismutated in a form of recessive amyotrophic lateral sclerosis,” Nat.Genet., 29(2): 160-65 (2001); Hadano, S., et al., “A gene encoding aputative GTPase regulator is mutated in familial amyotrophic lateralsclerosis 2,” Nat. Genet., 29(2): 166-73 (2001)). About 15-20% offamilial cases are due to mutations in the gene encoding Cu/Znsuperoxide dismutase 1 (SOD1) (Siddique, T., et al., “Linkage of a genecausing familial amyotrophic lateral sclerosis to chromosome 21 andevidence of genetic-locus heterogeneity,” N. Engl. J. Med., 324(20):1381-84 (1991); Rosen, D. R., et al., “Mutations in Cu/Zn superoxidedismutase gene are associated with familial amyotrophic lateralsclerosis.” Nature, 362(6415): 59-62 (1993)).

Although ALS is characterized by loss of motor neurons in the spinalcord resulting in muscle atrophy, the disease also manifests itself withchanges in protein folding, protein aggregation, and mitochondrialdysfunction. Early symptoms of ALS include increasing muscle weakness,particularly in the arms and legs, and in the muscles associated withspeech, swallowing and breathing. Symptoms of weakness and muscleatrophy usually begin asymmetrically and distally in one limb, and thenspread within the neuroaxis to involve contiguous groups of motorneurons. Symptoms can begin either in bulbar or limb muscles. Clinicalsigns of both lower and upper motor neuron involvement are required fora definitive diagnosis of ALS. Respiration is usually affected late inlimb onset patients, but occasionally can be an early manifestation inpatients with bulbar onset symptoms.

Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic disease that is characterized by“attacks,” during which areas of white matter of the central nervoussystem, known as plaques, become inflamed. Inflammation of these areasof plaque is followed by destruction of myelin, the fatty substance thatforms a sheath or covering that insulates nerve cell fibers in the brainand spinal cord. Myelin facilitates the smooth, high-speed transmissionof electrochemical messages between the brain, spinal cord, and the restof the body. Damage to the myelin sheath can slow or completely blockthe transmission of these electrochemical messages, which can result indiminished or lost bodily function.

The most common course of MS manifests itself as a series of attacks,which are followed by either complete or partial remission, during whichthe symptoms lessen only to return at some later point in time. Thistype of MS is commonly referred to as “relapsing-remitting MS.” Anotherform of MS, called “primary-progressive MS,” is characterized by agradual decline into the disease state, with no distinct remissions andonly temporary plateaus or minor relief from the symptoms. A third formof MS, known as “secondary-progressive MS,” starts as arelapsing-remitting course, but later deteriorates into aprimary-progressive course of MS.

The symptoms of MS can be mild or severe, acute or of a long duration,and may appear in various combinations. These symptoms can includevision problems such as blurred or double vision, red-green colordistortion, or even blindness in one eye, muscle weakness in theextremities, coordination and balance problems, muscle spasticity,muscle fatigue, paresthesias, fleeting abnormal sensory feelings such asnumbness, prickling, or “pins and needles” sensations, and in the worstcases, partial or complete paralysis. About half of the people sufferingfrom MS also experience cognitive impairments, such as for example, poorconcentration, attention, memory and/or judgment. These cognitivesymptoms occur when lesions develop in those areas of the brain that areresponsible for information processing.

Experimental autoimmune encephalomyelitis (EAE) is an experimentalautoimmune disease of animals that is thought to model aspects ofmultiple sclerosis (Zamvil et al. (1990) Annu. Rev. Immunol. 8:579-621). EAE can be induced in susceptible strains of rats, such as theLewis rat, by immunization to myelin basic protein (MBP) in completeFreund's adjuvant (CFA), an emulsion of mineral oil containing killedMycobacteria. The disease develops about 12 days after immunization andis characterized by paralysis of various degrees due to inflammation ofthe central nervous system. The paralysis can last up to 6 or 7 days andthe rats usually recover unless they die during the peak of their acuteparalysis. EAE is caused by T cells that recognize defined determinantsof the MBP molecule. The major MBP determinant in the Lewis rat iscomposed of the peptide sequence 71-90 (Zamvil et al. Supra).

Alternatively, in vitro cell lines for MS can also be used. Such invitro cell lines include, but are not limited to, the LM7PC and PLI-2cell lines. These two continuous cell lines were derived from humanchoroid plexus cells originating from two different patients sufferingfrom MS obtained by a culture method described in the documentWO-A-9320188 and U.S. Pat. No. 6,342,383 to Perron et al.

Alzheimer's Disease

Alzheimer's disease is a progressive, neurodegenerative disease thataffects the portions of the brain that control thought, memory andlanguage. This disease is characterized by progressive dementia thateventually results in substantial impairment of both cognition andbehavior. The disease manifests itself by the presence of abnormalextracellular protein deposits in brain tissue, known as “amyloidplaques,” and tangled bundles of fibers accumulated within the neurons,known as “neurofibrillary tangles,” and by the loss of neuronal cells.The areas of the brain affected by Alzheimer's disease can vary, but theareas most commonly affected include the association cortical and limbicregions. Symptoms of Alzheimer's disease include memory loss,deterioration of language skills, impaired visuospatial skills, andimpaired judgment, yet those suffering from Alzheimer's retain motorfunction.

Alzheimer's disease is characterized by two hallmark pathologicalfeatures that involve protein misfolding: Neurofibrillary tangles (NFTs)formed by paired helical filaments (PHFs) from abnormally modified Tauprotein and senile plaques composed of beta-amyloid (A.beta.) (SeePrice, et al., (1998) Annu Rev Neurosci 21: 479-505). Mild cognitiveimpairment (MCI) is observed in Alzheimer's disease and is thought torepresent the prodromal stage of Alzheimer's disease. MCI accompaniesneuronal loss in Alzheimer's disease. Dementia and neuronal loss inAlzheimer's disease correlate significantly with levels of Tau pathologyand resulting NFTs. Evidence for altered/reduced proteasomal activity inAlzheimer's disease has been found that may result from the defectiveubiquination and/or breakdown of misfolded proteins such as PHF-Tau andbeta amyloid by the 20S proteasome (Keck, et al. (2003) J Neurochem85:115-22; Keller et al. (2000) J Neurochem 75: 436-9; and Lopez et al.,(2003) Exp Neurol 180: 131-43). Additionally, a mutant form of ubiquitin(Ub+1), generated by molecular misreading, was observed in the brains ofAlzheimer's disease patients including those with the non-familialAlzheimer's disease (van Leeuwen, et al. (1998) Science 279: 242-7; andLam, et al., (2000) Proc Natl Acad Sci USA 97: 9902-6). Ub+1 cappedpolyUb chain was also able to inhibit proteasomal activity in vitro andmay induce accumulation of misfolded proteins and contribute to bothA.beta. and Tau pathology in Alzheimer's disease (Lam, et al., (2000)Supra).

Proteasomal dysregulation can lead to a variety of cellular alterationsthat can contribute to chronic neurodegeneration some of which includepolyamine dysregulation and cell cycle dysregulation, inflammation andapoptosis (See e.g., Jesenberger, et al. (2002) Nat Rev Mol Cell Biol 3:112-21; Li, et al. (2003) Int J Biochem Cell Biol 35: 547-52; Bernstein,et al. (1995) Neurosci Lett 186:123-6; and Trojanowski et al. (2000) AnnN Y Acad Sci 924: 62-7). Expression of cell cycle regulating geneproducts and induction of DNA replication (clear indications of cellcycle re-entry) has been demonstrated in Alzheimer's disease andParkinson's disease (Jordan-Sciutto, et al. (2002) J Neuropathol ExpNeurol 61: 358-67; Klein, et al. (2003) J Clin Invest 111: 785-93;Nouspikel, et al. (2003) Bioessays 25: 168-73; and Yang, et al. (2001) JNeurosci 21: 2661-8). Most recently Yang et al demonstrated that thecell cycle induction in Alzheimer's disease is observed during both theearly prodromal stage (MCI) and in the advanced stages of Alzheimer'sdisease indicating that neurons dwell in an unproductive cell cycle formany months before finally committing to apoptosis (Yang, et al. (2003)J Neurosci 23: 2557-63). The protective effect of flavopiridol, apan-CDK inhibitor, in a model of proteasome inhibition-induced neuronaldeath, together with the finding of cycling CDK induction in an in vitroA.beta. model of Alzheimer's disease demonstrate a link betweenproteasomal dysfunction and cell cycle dysregulation and neuronal death(Jordan-Sciutto, et al. (2001) Mech Ageing Dev 123: 11-20; and Rideout,et al. (2003) J Neurosci 23: 1237-45).

A suitable animal model for Alzheimer's disease that mimics thepathology of the disease in humans can be one in which a selectivelesion is placed in a subcortical nucleus (nucleus basalis of Meynert)with a resultant cortical cholinergic deficiency, similar in magnitudeto that seen in early to moderate stage Alzheimer's disease. Numerousbehavioral deficits, including the inability to learn and retain newinformation, are characteristic of this lesion. Pharmacological agentsthat can normalize these abnormalities would have a reasonableexpectation of efficacy in Alzheimer's disease (See e.g., Haroutunian,et al. (1985) Life Sciences, 37:945-952).

In addition to in vivo models, a number of in vitro cell lines can alsobe used to examine the effects of pharmacological agents on Alzheimer'sdisease such as apolipoprotein E uptake and low-density lipoproteinreceptor-related protein expression by the NTera2/D1 cell line, a cellculture model for late-onset Alzheimer's disease (See e.g., Williams etal. (1997) Neurobiol. of Disease, 4:58-67). Alternatively, humanmelanocytes can be used as a model system for studies of Alzheimer'sdisease (See e.g., Yaar et al. (1997) Arch. Dermatol. 133:1287-291).

Parkinson's Disease

Parkinson's disease is a motor system disorder caused by the loss ofnerve cells, or neurons, found in the sub stantia nigra region of themid-brain. These neurons produce dopamine, a chemical messenger moleculethat is found in the brain and helps control or direct muscle activity.Dopamine is used by the cells of the substantia nigra as aneurotransmitter to signal other nerve cells. Parkinson's disease occurswhen these neurons die or become impaired, thereby decreasing dopaminelevels within the brain. Loss of dopamine causes the neurons to fireuncontrollably, which leaves patients unable to direct or control theirbodily movement in a normal manner. The four main symptoms ofParkinson's disease are trembling in the hands, arms, legs, jaw andface; stiffness of the limbs and/or trunk; a slowness of movement,referred to as bradykinesia; and impaired balance and/or coordination.Parkinson's disease is both chronic, i.e., it persists over a longperiod of time, and progressive, i.e., the symptoms grow worse overtime.

Animal models of Parkinson's disease are well established, such as theprimate model of Parkinson's Disease described by Zamir et al. (1984)Brain Res. 322, 356-60. Neurodegenerative disease-causing substance canbe used to cause a neurodegenerative disease in a mammal. Examples ofsuch substances include N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), 1-methyl-4-henylpyridine (MPP.sup.+) and manganese dust forParkinson's disease; quinolinic acid for Huntington's chorea; andbeta-N-methylamino-L-alanine for amyotrophic lateral sclerosis,Parkinson's disease and Alzheimer's disease. Due to their mimicry ofeffects of Parkinson's disease, treatment of animals withmethamphetamine or MPTP has been used to generate models for Parkinson'sdisease. The end result of MPTP administration is the destruction of thestriatum in the brain, an area in the neocortex limbic system in thesubcortical area in the center of the brain, an area compromised inParkinson's disease. The neurotransmitter dopamine is concentrated inthe striatum Parkinson's disease is characterized by lesions in thatarea of the brain and by depleted dopamine levels. In some species(primates) the striatal degeneration has been reported to be accompaniedby behavioral symptoms that mimic Parkinson's symptoms in humans (Seee.g., Markey, et al. (1986) Medicinal Research Reviews 6:389-429).

Huntington's Disease

Huntington's disease is a hereditary disorder caused by the degenerationof neurons in certain areas of the brain. This degeneration isgenetically programmed to occur in certain areas of the brain, includingthe cells of the basal ganglia, the structures that are responsible forcoordinating movement. Within the basal ganglia, Huntington's diseasespecifically targets nerve cells in the striatum, as well as cells ofthe cortex, or outer surface of the brain, which control thought,perception and memory. Neuron degeneration due to Huntington's diseasecan result in uncontrolled movements, loss of intellectual capacity andfaculties, and emotional disturbance, such as, for example, mood swingsor uncharacteristic irritability or depression.

As discussed above, neuron degeneration due to Huntington's disease isgenetically programmed to occur in certain areas of the brain. Studieshave shown that Huntington's disease is caused by a genetic defect onchromosome 4, and in particular, people with Huntington's disease havean abnormal repetition of the genetic sequence CAG in the Huntington'sdisease gene, which has been termed IT15. The IT15 gene is located onthe short arm of chromosome 4 and encodes a protein called huntingtin.Exon I of the IT15 gene contains a polymorphic stretch of consecutiveglutamine residues, known as the polyglutamine tract (Rubinsztein,(2002) TRENDS in Genetics, 18: 202-9). Asymptomatic individualstypically contain fewer than 35 CAG repeats in the polyglutamine tract.Murine models for HD include that described by Hayden et al. in U.S.Pat. No. 5,849,995, as well as in vitro systems as described in U.S.Pat. No. 5,834,183 to Orr et al.

Prion-Associated Diseases

The prion protein (PrP) is closely associated with a group of fatalneurodegenerative diseases (Ma, et al. (2001) Proc. Natl. Acad Sci.,98:14955-14960). This group of disorders is characterized by vacuolationof the brain's gray matter, also known as spongioform change. Thesediseases can take a variety of forms. For example, these diseases can besporadic, dominantly heritable, as well as transmissible disorders. Inhumans, the most prevalent form of prion disease is Creutzfeldt-Jakobdisease, while in animals, the most common form is known as scrapie.Other disorders in this group include kuru,Gerstmann-Straussler-Scheinker disease and fetal familial insomnia. Alldisorders are invariably fatal.

In particular, the symptoms of Creutzfeldt-Jakob disease include arapidly progressive deterioration of intellectual abilities (also knownas dementia). The median duration of this illness, from on-set ofsymptoms to death is around four months. As the disease stateprogresses, the dementia is typically accompanied by other symptoms suchas ataxia, muscular rigidity, and spontaneous and irregular limb jerks,also known as myoclonus.

Spinocerebellar Ataxia

Ataxias are diseases wherein a person loses the ability to coordinatemuscle activity during voluntary muscle contraction, and therefore,loses the ability to coordinate smooth bodily movements. Spinocerebellarataxia is the most common form of hereditary ataxia. Symptoms of theon-set of spinocerebellar ataxia include limb ataxia, nystagmus(rhythmical oscillation of the eyeballs, in either a pendular or jerkymotion), kyphoscoliosis (a deformity of the spine characterized byextensive flexion), and pes cavus (a contracted foot, or exaggeration ofthe normal arch of the foot). The major pathological changes that occurwith the disease state occur in the posterior columns of the spinalcord. Spinocerebellar ataxia is most often an autosomal recessiveinherited disorder.

Among the adult-onset dominant spinocerebellar ataxias (SCAs), sevendifferent loci have been mapped (Gispert et al. (1993) Nature Genet. 4,295-299; Takiyama et al. (1993) Nature Genet. 4, 300-304; Gardner et al.(1994) Neurology, 44: A361; Nagafuchi et al. (1994) Nature Genet. 6:14-18; Ranum et al. (1994) Nature Genet. 8, 280-284; Benomar et al.(1995) Nature Genet. 10: 84-88; Gouw et al. (1995) Nature Genet. 10:89-93; Zhuchenko et al. (1997) Nature Genet. 15: 62-69). Approximatelysixty percent of the dominant ataxias result from expansions intrinucleotide CAG repeats at the SCA1, 2, 3, 6 or 7 loci (Nagafuchi etal. (1994) Nature Genet. 6: 14-18; Zhuchenko et al. (1997) Nature Genet.15: 62-69; Orr et al. (1993) Nature Genet. 4: 211-226; Kawaguchi et al.(1994) Nature Genet. 8: 221-228; Koide et al. (1994) Nature Genet. 6:9-13; Imbert et al. (1996) Nature Genet. 14: 285-291; Pulst et al.(1996) Nature Genet. 14: 269-276; Sanpei et al. (1996) Nature Genet. 14:277-284; David et al. (1997) Nature Genet. 17: 65-70; Koob et al. (1998)Nature Genet. 18: 72-75. The substantial clinical variability among theremaining 40% of the genetically undefined dominant families suggeststhat a number of additional ataxia coding sequences remain to beidentified. Suitable models are, for example the SCAT murine modeldisplaying neurodegeneration with progressive ataxin-7 accumulation (Seee.g. Yvert et al. (2001) Hum Mol Genet. 10:1679-92), as well as in vitrosystems as described in U.S. Pat. No. 5,834,183 to Orr et al.

Spinal Muscular Atrophy

Spinomuscular atrophy (SMA) is a disease of the anterior horn cells ofthe spinal cord. There are several different types of SMA, includingType I or Acute (Severe) SMA, which is also known as Werdnig-HoffmannDisease, Type II (Chronic) SMA, Type III (Mild) SMA, often referred toas Kugelberg-Welander or Juvenile SMA, Type IV (Adult Onset) SMA, andAdult Onset X-Linked SMA, also known as Kennedy's Syndrome orBulbo-Spinal Muscular Atrophy, which occurs in males, but females may becarriers. SMA affects the voluntary muscles that are responsible foractivities such as crawling, walking, head and neck control, andswallowing. SMA mainly affects the proximal muscles, or the musclescloses to the trunk of a person's body. Symptoms include weakness in thelegs and arms, with weakness in the legs being greater than weakness inthe arms. Other symptoms may include tongue fasciculations, or abnormalmovements of the tongue. During the course of SMA, however, a person'ssenses, feelings and intellectual activity remain unaffected.

Suitable animal models of spinal muscular atrophy include, but are notlimited to, the murine models described Fricker, (2000) Drug DiscoveryToday 5:220-221; Frugier, et al. (2000) Human Molecular Genetics9:849-858; Hsieh-Li, et al. (2000) Natural Genetics 24:66-70; andMonani, et al. (2000) Human Molecular Genetics 9:333-339. In vitrosystems of spinal muscular atrophy can be those described by Yoshida, etal. (1990) J. Biol. Chem. 265:17174-17179.

Endoplasmic Reticulum (ER) Stress

Endoplasmic reticulum is an organelle responsible for folding,post-translational modifications and transport of secretory, luminal andmembrane proteins. This organelle, therefore, plays an important role inmaintaining cellular homeostasis. Endoplasmic reticulum (ER) stress is acondition that is accelerated by accumulation of unfolded or misfoldedproteins after the disturbance of the ER environment, which can betriggered by a variety of physiological and pathological factors, suchas nutrient deprivation, altered glycosylation, calcium depletion,oxidative stress, DNA damage and energy disturbance. The ER stress mayinitiate the unfolded protein response (UPR) to restore cellularhomeostasis or lead to apoptosis.

At the early stages of ER stress, the failure of molecular chaperonesbreak the maintenance of proteostasis (Kelly et al., 2007; Naido et al.,2009). To overcome the deleterious effects of ER stress, a series ofadaptive and protective strategies are initiated. For example, owing tothe abundance of unfolded or misfolded proteins accumulated in the ER,new protein synthesis is inhibited. To clear the accumulated proteins,many chaperone genes are induced at the transcriptional level andactivate the ER-associated degradation (ERAD) system, which cantranslocate and remove misfolded proteins through proteasomaldegradation. These processes are identified as the unfolded proteinresponse (UPR) (Meusser et al., 2005; Lynch et al., 2012). However, ifunresolved, ER stress can be lethal to cells via what is recognized asER stress induced apoptosis (Zeng et al., 2015).

The UPR is a concerted and complex cellular response that is mediatedthrough three ER transmembrane receptor proteins, such as thedouble-stranded RNA-activated protein kinase like endoplasmic reticulumkinase (PERK), inositol-requiringenzyme 1 (IRE1, also called ERN1) andactivating transcription factor 6 (ATF6) (Promlek et al., 2011; Teske etal., 2011).

The ER transmembrane receptor protein PERK is a member of the eukaryotictranslation initiation factor 2a (eIF2a) kinase subfamily, together withRNA-dependent protein kinase (PKR), general control nondepressible 2kinase (GCN2) and heme-regulated eIF2a kinase (HRI) (Hardin et al.,1999). PERK possesses a luminal domain like IRE1 and a cytoplasmicportion that manifests protein serine/threonine kinase activity, and ithas a PKR-like catalytic domain, which most importantly phosphorylateseIF2α (Shi et al., 1998). eIF2 has three subunits, i.e., α, β, and γ,and eIF2α is a translation initiation factor that functions in the earlysteps of protein synthesis by forming a ternary complex with GTP andinitiator tRNA. This complex binds to a 40S ribosomal subunit, followedby mRNA binding to constitute a 43S preinitiation complex. Junction ofthe 60S ribosomal subunit to form the 80S initiation complex is precededby hydrolysis of the GTP bound to eIF2 and release of an eIF2-GDP binarycomplex. In order to make eIF2 recycle and then to catalyze anotherround of initiation, the GDP bound to eIF2 typically must exchange withGTP by way of the reaction catalyzed by eIF2β. Here, eIF2αphosphorylation is key to stabilize the eIF2/GDP/eIF2β complex andprevent the GDP/GTP exchange reaction. Therefore, eIF2α phosphorylationimpairs the recycling of eIF2 between successive rounds of initiationand leads to global inhibition of translation at early stages of ERS.

Because ER stress plays an essential role in many diseases, such asneurodegenerative disease like amyotrophic lateral sclerosis, drugstargeting ER stress and its different actors are good candidates forpossible cures. Several drugs and natural products have been proposed tohinder ER stress such as Salubrinal.

Dephosphorylation Inhibitors

Salubrinal, a cinnamarnide derivative, has been shown to prevent eIF2αdephosphorylation by inhibiting the protein complex GADD34/proteinphosphatase 1 (PP1), which consists of the general cellularserine/threonine phosphatase PP1 and the non-enzymatic cofactor GADD34(Boyce et al., 2005; Long et al., 2005). As discussed above, the eIF2αphosphorylation appears to be cytoprotective during ER stress byinhibiting the translation initiation activity of eIF2a, which reducesglobal protein synthesis and results in a reduction of the ER workload(Hotamisligil et al., 2010). For instance, activating transcriptionfactor 4 (ATF4), a transcription factor that induces the expression ofUPR target genes, is produced through alternative translation and thusnot inhibited by phosphorylation of eIF2α (Iniga et al., 2010; andHotamisligil et al., 2010).

Salubrinal appears to be a candidate compound for cytoprotection againstendoplasmic reticulum stress and the unfolded protein response thatleads to numerous diseases. However, it is not an ideal candidate toprevent eIF2α dephosphorylation because of its low solubility in aqueoussolutions, rapid clearance and relatively high toxicity in animals(EC50˜15 μM).

Salubrinal Analogs

A class of dephosphorylation inhibitors of eIF2-alpha according to theinvention can be defined by the compounds of Formula (II), Salubrinalanalogs (excluding Salubrinal itself):

In some embodiments, R¹ is H, C₃₋₆ cycloheteroalkyl, or N(CH₃)(CH₃), R²is H, C₃₋₆ cycloheteroalkyl, or when combined with R⁴ is together—CH(C₁₋₅heteroalkyl)-, R³ is H, CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆cycloheteroalkyl, amino acid, amino acid derivative, or C₁₋₁₀heteroalkyl, R⁴ is null, H, CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆cycloheteroalkyl, amino acid, amino acid derivative, or C₁₋₁₀heteroalkyl, R⁵ is ═S, CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆cycloheteroalkyl, amino acid, amino acid derivative, or C₁₋₂₀heteroalkyl, A-B is CH—CH₂ or CH═CH, and D-E is C—N, CH—N, or C═N.

Examples of compounds according to the Formula (II) are provided intables I and II below. Each of the compounds in these table has apotency to inhibit, for example and without limitation, thedephosphorylation of eIF2-alpha.

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

TABLE 2 ID Structure Formula M.W. 1901

C24H21Cl3N4O3S 551.87 1902

C27H27Cl3N4O3S 593.95 1903

C24H21Cl3N4O2S 535.87 1904

C28H27Cl3N4O2S 589.09 1905

C26H25Cl3N4O2S 563.93 1906

C25H23Cl3N4O3S 565.9 1907

C29H29Cl3N4O3S 619.99 1908

C35H25Cl3N4O3S 688.02 1909

C28H21Cl3N4O2S 583.92 1910

C26H25Cl3N4O3S 579.93 1911

C29H23Cl3N4O3S 613.94 1912

C25H23Cl3N4O4S 581.9 1913

C30H31Cl3N4O3S 634.02 1914

C28H29Cl3N4O4S 623.98 1915

C26H25Cl3N4O2S 563.93 1916

C24H21Cl3N4O3S 551.87 1917

C29H29Cl3N4O2S 603.99 1918

C28H27Cl3N4O4S 621.96 1919

C27H29Cl4N5O3S 608.97 1920

C28H231Cl4N5O3S 622.99 1921

C28H29Cl3N4O3S 607.98 1922

C24H21Cl3N4O4S 567.87 1923

C26H25Cl3N4O4S 595.93 1924

C27H27Cl3N4O4S 609.95 1925

C27H27Cl3N4O4S 609.95 1926

C33H37Cl3N4O3S 676.1 1927

C33H29Cl3F3N5O5S 657.01 1928

C32H35Cl3N4O3S 662.07 1929

C30H33Cl3N4O3S 636.03 1930

C31H35Cl3N4O3S 650.06 1931

C27H26Cl4N5O3S 606.95 1932

C31H32Cl3N5O6S 709.04 1933

C29H29Cl3N4O5S 651.99 1934

C28H27Cl3N4O5S 637.96 1935

C27H25Cl3N4O5S 623.94 1936

C30H31Cl3N4O5S 666.01 1937

C32H34Cl3N5O6S 723.07 1938

C31H30Cl3N5O6S 707.02 1939

C35H32Cl3N5O6S 757.08 1940

C31H33Cl3N4O5S 680.04 1941

C30H31Cl3N4O5S 666.01 1942

C22H19Cl3N4OS 493.84 1943

C27H27Cl3N4O5S 625.95 1944

C26H25Cl3N4O3S 579.93 1945

C32H35Cl3N4O5S 694.04 1946

C30H31Cl3N4O5S 666.01 1947

C33H37Cl3N4O5S 708.09 1948

C34H38Cl3N5O6S 751.12 1949

C28H30Cl3N5O4S 638.99

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (III), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (IV), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (V), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (VI), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (VII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (VIII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (IX), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (X), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (XI), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (XII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In one embodiment, a phosphorylation inhibitors of eIF2-alpha accordingto the invention has the Formula (XIII), as shown below,

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, groups R¹, R², R³, R⁴ and R⁵ from Formula (II)correspond to sites available to link a functional group (e.g., apro-drug group) and to generate a Salubrinal derivative. For example,without limitation, site R¹ can be linked to a functional group via aMichael addition/beta-elimination and sites R²⁻⁵ can be linked to afunctional group via an alkylation/regeneration through enzymatic orchemical cleavage. In another example, without limitation, site R³ canbe linked to a functional group by reacting a 1-chloroamide with anucleophile. In another example, without limitation, site R⁴ can belinked to a functional group by reacting a thioisocyanate with anucleophile. In another example, without limitation, site R⁴ can belinked to a functional group by acylation of salubrinal withacylchloride. In another example, without limitation, site R⁵ can belinked to a functional group by the alkylation of salubrinal withchloroacetals. In another example, without limitation, sites R³ and R⁴can be linked to a functional group by using a hemi-aminal and theenzymatic cleavage of the hemi-aminal capping group.

In some embodiments, groups R¹, R², R³, R⁴, and R⁵ from Formula (II) areamino acid or amino acid derivatives. The amino acid can be, forexample, alanine (ala—A), arginine (arg—R), asparagine (asn—N), asparticacid (asp—D), cysteine (cys—C), glutamine (gln—Q), glutamic acid(glu—E), glycine (gly—G), histidine (his—H), isoleucine (ile—I), leucine(leu—L), lysine (lys—K), methionine (met—M), phenylalanine (phe—F),proline (pro—P), serine (ser S), threonine (thr—T), tryptophan (trp—W),tyrosine (tyr—Y), valine (val—V), or any combinations thereof.

A “derivative” or “analog” is defined as a eIF2-alpha phosphorylationinhibitor that has been subjected to chemical modification.Derivatization may include the substitution of certain chemical groupsto Salubrinal. Such derivatizations are known from the state of the art.The derivatives and analogs maintain the biological activity ofSalubrinal and function in a similar manner, but can offer advantages tothe compound, such as a longer life, resistance to decomposition orincreased activity. The term “analog” encompasses derivatives ofsalubrinal as well as chemically synthesized molecules sharing certainchemical similarities to salubrinal.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol, and the like. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate. Compounds described herein are optionally invarious forms, including but not limited to, amorphous forms, milledforms and nano-particulate forms. In addition, compounds describedherein include crystalline forms, also known as polymorphs. Polymorphsinclude the different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate. The compounds described herein may be incrystalline or amorphous form, and may be in the form of a hydrate or asolvate. Several polymorphs of the crystalline form can be formulatedand are also encompassed within the invention.

EXAMPLES

Aspects of the applicant's teachings may be further understood in lightof the following examples, which should not be construed as limiting thescope of the applicant's teachings in any way or by necessarilyindicative to the optimal ways that the invention can be practice.

Synthesis of Salubrinal Analog—Compound ID 1912

By way of example, a description of the synthesis for compound ID 1912is presented in FIG. 1 . The sequence of the synthesis consists of 7distinct chemical product structures and involves chromatography.Compound ID 1912 was produced with a purity of 98.2% in 7 steps with anoverall yield of 13% from cinnamamide as the starting material. Alloxygen and/or moisture sensitive reactions were carried out under N₂atmosphere. All reagents and solvents were purchases from commercialvendors and used as received. 1H NMR spectra were recorded on a 500 MHzspectrometer. HPLC conditions for all LCMS on Waters Alliance 2695reported: Waters XTerra RP18 34.6×30 mm, 3.5 μm.; hold 5% B for 0.2minute, 5% to 100% B in 1.8 minutes, then hold 100% B for 1.0 minute,run time=3.0 min; Eluents: A 10 mM NH4COOH in water; B=MeCN.

Step 1.

Cinnamamide and chloral hydrate were dissolved in toluene; the mixturewas heated to 120° C. The RBF was left open for 20 min, to allow waterand toluene co-evaporates. The condenser was applied and the reactionwas stirred for 36 hrs. The mixture was concentrated, and the residuewas dissolved in 50 mL EtOAc, and reflux for 2 hrs, the cool the mixtureto room temperature, and filter, yielding 3.9052 g white crystal. 1HNMR: (500 MHz, CDCl3) δ 7.68 (d, J=15.6 Hz, 1H), 7.49-7.43 (m, 2H),7.35-7.29 (m, 3H), 6.38 (d, J=15.6 Hz, 1H), 6.27 (d, J=8.8 Hz, 1H), 6.02(dd, J=9.1, 5.1 Hz, 1H), 3.87 (s, 1H).

Step 2.

Alcohol was dissolved in 40 mL dry THF. To this solution, 4 mL SOCl2 wasadded, after the addition, the mixture was heated to 60° C. for 3 hrs.Then the solvent and excess SOCl2 was rotavapped, the residue was usedas crude for the next step.

Step 3.

Crude residue from reaction 229-17 was dissolved in acetone, and to thesolution was added 2.7 g KSCN. The mixture was stirred at reflux for 2hrs. After cooling down to room temperature, the mixture was filtered toremove inorganic salt. Then the filtrate was concentrated, and EtOAc wasadded to precipitate the inorganic by-product, and the slurry wasfiltered again. Concentrated the filtrate, purified the residue by ISCO,20-30% EtOAc/Hexane, 10% EtOAc/Hexane to load the solid, yielding lightyellow solid 3.61 g.

Step 4.

Isothiocynate and quinoline were dissolved in THF and heated up at 60°C. for 1 h. Then solvent was evaporated, and 50 mL DCM was added toprecipitate the product. The slurry was filtered, yielding 4.8328 g grayproduct.

Step 5.

Add acyl chloride and paraformaldehyde to dry ZnCl2 in dry RBF. Thereaction is stirred at 110° C. for 1.5 h and then diluted by water andextracted by Et2O. The combined organic layers were dried andconcentrated. The resulting residue was distilled, and the product comesout at around 40-50° C. (normal pump, the pressure is not clear),yielding 0.92 g clear liquid. 1H NMR: (500 MHz, CDCl3) δ 5.79 (s, 2H),4.13 (s, 2H), 3.50 (s, 3H).

Step 6.

Chloride was dissolved in 8 mL acetone, and to the solution was addedNaI. The reaction was stirred at room temperature for 2 hrs. Acetone wasremoved by rotavap, and 5 mL DCM was added to crash out the inorganicsalt, and then filtered the mixture. The filtrate was rotavaped toremove DCM, the crude iodide was used in next step as crude.

Step 7.

Salubrinal was suspended in 40 mL ACN (more dilute than 229-45). To themixture was added DIPEA and iodide. The reaction was stirred at Roomtemperature. LCMS showed 29% product. The mixture was filtered torecover salubrinal, washed with ACN. The filtrate was concentrated andpurified by reverse-combiflash (20% ACN/water 5 min, 20-55% ACN/water 5min, 55-75% 15 min, the compound came out at 66%). The collectedsolution was concentrated and lyoed, yield 263.2 mg white product, 98.2%pure. 1H NMR: (500 MHz, DMSO) δ 9.74 (s, 1H), 9.05 (d, J=8.7 Hz, 1H),8.95-8.91 (m, 2H), 8.45 (dd, J=8.3, 1.6 Hz, 1H), 7.70-7.64 (m, 2H),7.61-7.55 (m, 4H), 7.46-7.38 (m, 3H), 6.88 (d, J=15.8 Hz, 1H), 6.34 (d,J=8.8 Hz, 1H), 5.85-5.74 (m, 2H), 4.36-4.24 (m, 2H), 3.35 (s, 3H).

Synthesis of Salubrinal Analog—Compound ID 1913

By way of example, a description of the synthesis for compound ID 1913is presented in FIG. 2 . The sequence of the synthesis consists of 7distinct chemical product structures and involves chromatography.Compound ID 1913 was produced with a purity >99% in 7 steps with anoverall yield of 12% from cinnamamide as the starting material. Alloxygen and/or moisture sensitive reactions were carried out under N2atmosphere. All reagents and solvents were purchases from commercialvendors and used as received. 1H NMR spectra were recorded on a 500 MHzspectrometer. HPLC conditions for all LCMS on Waters Alliance 2695reported: Waters XTerra RP18 34.6×30 mm, 3.5 μm.; hold 5% B for 0.2minute, 5% to 100% B in 1.8 minutes, then hold 100% B for 1.0 minute,run time=3.0 min; Eluents: A 10 mM NH4COOH in water; B=MeCN.

Step 1.

Compound 1 (3.0 g, 20.4 mmol) and chloral hydrate (4.7 g, 28.5 mmol)were dissolved in toluene (150 mL) and the mixture was stirred at 120°C. overnight. The reaction was then concentrated in vacuum. To themixture was added EtOAc (50 mL) and refluxed for another 2 hours. Filterthe precipitate and obtained compound 2 (5.3 g, 89% in yield).

Steps 2-3.

To a solution of compound 2 (5.3 g, 18.0 mmol) in THF (50 mL, dry) wereadded thionyl chloride (5.0 mL, 68.9 mmol). The resulting reactionmixture was stirred at 60° C. for 3 hrs under nitrogen atmosphere. Thereaction mixture was concentrated in vacuum to give crude compound 3which was dissolved in acetone (50 mL). To the mixture was added KSCN(3.5 g, 36.1 mmol). The resulting reaction mixture was refluxed for 2hours and cooled to room temperature. Precipitate was filtered off andthe filtrate was concentrated in vacuum. Dilute the crude was EtOAc (30mL) and filter off the precipitate again. The filtrate was concentratedin vacuo and compound 4 (4.3 g, 72%) was obtained after flash columnchromatography (0% EtOAc in hexanes to 50% EtOAc in hexanes).

Step 4.

To THF (50 mL) was added compound 4 (4.3 g, 12.8 mmol) and8-aminoquinoline sequentially. The mixture was stirred at 60° C. for 1h. The solvent was removed in vacuUM and to the mixture as added DCM (50mL) and stirred for 10 min. Filter the precipitate and Compound 5 (5.3g, 86% in yield) was obtained.

Step 5.

Compound 6 (3.6 mL, 24.5 mmol) was dissolved in DCM (50 mL) and themixture was cooled to 0° C. under N2 protection. To the mixture wasadded zinc chloride (0.4 g, 2.4 mol, anhydrous) followed by compound 7(2.4 mL, 37.0 mmol). The reaction is stirred at room temperature for 4hours and concentrated. The crude was diluted with Et2O/water (30 mL/30mL). The organic layer was separated, dried over Na2SO4 andconcentrated. Compound 8 (1.5 g, 29% in yield) was obtained after vacuumdistillation.

Steps 6-7.

Compound 8 (800 mg, 4.2 mmol) was dissolved in acetone (10 mL) and tothe solution was added NaI (784 mg, 5.2 mmol). The reaction is stirredat room temperature for 2 hours and the solvent in removed in vacuum.Then DCM (20 mL) was added and the precipitate was filtered off. Thefiltrate was concentrated in vacuo to give crude compound 9. Compound 9was added to a pre-stirred solution of compound 5 (1.0 g, 2.1 mmol) andDIPEA (809 mg, 5.2 mmol) in MeCN (20 mL). The reaction mixture isstirred at 60° C. for 16 hours. The solvent was removed in vacuo andcompound 10 (276 mg, 21% in yield) was obtained after flash columnchromatography (two purification is required, 1st purification is normalphase with silica column, from 0% EtOAc in hexanes to 30% EtOAc inhexanes. 2nd purification is C18 column, from 50% MeCN in 10 mM AmF inwater to 100% MeCN).

LC/MS/MS Characterization of Salubrinal Analogs

A description of the characterization of compound IDs 1901-1948 ispresented in FIG. 3 . Samples were analyzed by LC/MS/MS using a CTC PALautosampler, Shimadzu Prominence HPLC and an AB SCIEX API 4000 QTRAPtriple quadrupole mass spectrometer. The [M+H]⁺ adducts of the prodrugs,controls and internal standards were monitored using positive modeelectrospray ionization in MRM (multiple reaction monitoring) mode. Theanalytes were injected onto a C18 column (HyPURITY 50×2.1 mm, 3μ) andchromatographed using a reverse phase gradient with 0.1% formic acid inwater and 0.1% formic acid in acetonitrile mobile phases.

Stability of Salubrinal Analogs in Phosphate Buffer and Mouse Plasma

A description of the stability of compound IDs 1901-1931 in phosphatebuffer and mouse plasma is presented in FIG. 4A. A description of thestability of compound IDs 1932-1948 in phosphate buffer and mouse plasmais presented in FIG. 4B. A description of the stability of two referencecompounds, i.e., salubrinal and diltiazem, in phosphate buffer and mouseplasma is presented in FIG. 4C.

The test articles for the mouse plasma stability test were incubated induplicate in mouse plasma at 370° C. for 60 minutes. Non-enzymaticdegradation of the prodrug was assessed in parallel incubations(duplicates) in 100 mM potassium phosphate buffer (pH 7.4). At the endof the incubation period, reactions were stopped by the addition of 4volumes of ice-cold stop solution (1 μM glyburide in acetonitrile) intoeach assay tube. Tubes were mixed thoroughly and centrifuged to removeprecipitated proteins. Resulting supernatants were collected (100 μL)and diluted (1:1) with LC/MS/MS mobile phase. The samples were analyzedby LC/MS/MS to determine the remaining parent and appearing drug(salubrinal).

Initial concentration of each prodrug in plasma and phosphate buffer wasdetermined in a separated well. Prodrug was added to premixed matrix andstop solution and processed as described above. Data are reported asmean % of parent (or prodrug) loss and % of total salubrinal (or drug)appearance.

The experimental conditions for the mouse plasma stability test were thefollowing:

TABLE 3 Mouse Plasma Stability Experimental Conditions Test Conc. 1 μMMatrices 100 mM Phosphate buffer, pH 7.4 and female CD1 mice plasmaIncubation 60 minutes at 37° C. Reference Compounds Salubrinal andDiltiazem

For the compounds salubrinal and diltiazem, the results were assessed induplicate as reference compounds.

Stability of Salubrinal Analogs in Mouse Liver Microsomes

A description of the stability of compound IDs 1901-1931 in mouse livermicrosomes is presented in FIG. 5A. A description of the stability ofcompound IDs 1932-1948 in mouse liver microsomes is presented in FIG.5B. A description of the stability of three reference compounds, i.e.,salubrinal, verapamit, and diltiazem, in mouse liver microsomes ispresented in FIG. 5C.

The article for the mouse liver microsomes stability test was incubatedin duplicate with microsomes at 370° C. for 0 and 45 minutes. Thereaction contained mouse liver microsomal protein (0.5 mg/mL) in 100 mMpotassium phosphate buffer with 1 mM NADPH at pH 7.4. Controlincubations (duplicates) were run for each test compound omitting NADPHto detect NADPH-free degradation. At the indicated time points, analiquot (40 uL) was removed from each experimental and control reactionand mixed 1:4 with ice-cold stop solution (1 μM labetalol inacetonitrile). The samples were centrifuged to remove precipitatedprotein, and the supernatants were further diluted with 1 volume ofLC/MS/MS mobile phase. The samples were analyzed by LC/MS/MS todetermine the remaining parent. Data are reported as mean % of parentloss.

The experimental conditions for the mouse liver microsomal stabilitytest was the following:

TABLE 4 Mouse Liver Microsomal Stability Experimental Conditions TestConc. 1 μM Matrices 100 mM Phosphate buffer, pH 7.4 and female CD1 mouseLC Protein Concentration 0.5 mg/mL Incubation 45 minutes at 37° C.Reference Compounds Diphenhydramine, Verapamil and Salubrinal

For the compounds salubrinal, verapamil, and diphenhydramine, theresults were assessed in duplicate as reference compounds.

Effect of Salubrinal Analogs on Cell Viability

A description of the cell viability in the absence of tunicamycin forcompound ID 1934 is presented in FIG. 6A. The dose of the compound ID1934 is varied between 0 μM and 30 μM, for each dose the test is done intriplicate, the mean viability is reported, the relative viability isreported, and area change is reported, and the total area change isreported. The area change can be plotted by compound dose, or summedover all tested doses, to compare compounds.

A description of the cell viability in function of the concentration ofcompound ID 1934 in the absence of tunicamycin is presented in FIG. 6B.The area of the curve is highlighted in gray.

A description of the cell viability area in function of theconcentration of compound IDs 1901-1948 and salubrinal controls Sal 1-7and Salu in the absence of tunicamycin is presented in FIG. 7 . The doseof the compounds and standards are varied between 0 μM and 30 μM.

A description of the total cell viability area change for compound IDs1901-1948 and salubrinal controls Sal 1-7 and Salu in the absence oftunicamycin is presented in FIG. 8 .

A description of the cell viability in the presence of tunicamycin forcompound ID 1934 is presented in FIG. 9A. The dose of the compound ID1934 is varied between 0 μM and 30 μM, the dose of the tunicamycin (TUN)is varied between 0 μM and 1 μM, for each dose the test is done intriplicate, the mean viability is reported, the relative viability isreported, and area change is reported, and the total area change isreported. The area change can be plotted by compound dose, or summedover all tested doses, to compare compounds.

A description of the cell viability in function of the concentration ofcompound ID 1934 in the presence of tunicamycin is presented in FIG. 9B.The area of the curve for each concentration of the compound ishighlighted in gray or black.

A description of the cell viability area in the presence of tunicamycinfor compound IDs 1901-1948 and salubrinal controls Sal 1-7 and Salu ispresented in FIG. 10 . The dose of the compounds and control samples arevaried between 0 μM and 30 μM.

A description of the total cell viability area change in the presence oftunicamycin for compound IDs 1901-1948 and salubrinal controls Sal 1-7and Salu is presented in FIG. 11 .

A description of the cell viability area in the absence of tunicamycinfor compound ID 1949 and salubrinal control Salub is presented in FIG.12 .

A description of the total cell viability area change in the absence oftunicamycin for compound ID 1949 and salubrinal control Salub ispresented in FIG. 13 .

A description of the cell viability area in the presence of tunicamycinfor compound ID 1949 and salubrinal control Salub is presented in FIG.14 .

A description of the total cell viability area change in the presence oftunicamycin for compound ID 1949 and salubrinal control Salub ispresented in FIG. 15 .

A description of the cell viability at different concentrations oftunicamycin for compound IDs 1901, 1906, 1912, and 1913 and salubrinalSalub is presented in FIG. 16 . The dose of the compounds and controlsamples are varied between 0 μM and 30 μM. The results are reportedafter 24 hrs. and 48 hrs. of cell grow concentration and after 30 minand 60 min of WST-1 development.

A description of the cell viability at different concentrations oftunicamycin without compound or salubrinal is presented in FIG. 17 . Theresults are reported after 24 hrs. and 48 hrs. of cell growconcentration and after 30 min and 60 min of WST-1 development.

A description of the cell viability in the absence of tunicamycin forcompound IDs 1901, 1906, 1912, and 1913 and salubrinal Salub ispresented in FIG. 18 . The results are reported after 24 hrs. and 48hrs. of cell grow concentration and after 30 min and 60 min of WST-1development.

A description of the cell viability at different concentrations oftunicamycin for compound IDs 1914, 1918, 1924, and 1946 and salubrinalSalub is presented in FIG. 19 . The dose of the compounds and controlsamples are varied between 0 μM and 30 μM. The results are reportedafter 24 hrs. and 48 hrs. of cell grow concentration and after 30 minand 60 min of WST-1 development.

A description of the cell viability at different concentrations oftunicamycin without compound or salubrinal is presented in FIG. 20 . Theresults are reported after 24 hrs. and 48 hrs. of cell growconcentration and after 30 min and 60 min of WST-1 development.

A description of the cell viability in the absence of tunicamycin forcompound IDs 1914, 1918, 1924, and 1946 and salubrinal Salub ispresented in FIG. 21 . The results are reported after 24 hrs. and 48hrs. of cell grow concentration and after 30 min and 60 min of WST-1development.

A description of the cell viability at different concentrations oftunicamycin for compound ID 1949 and salubrinal Salub is presented inFIG. 22 . The dose of the compound and control sample are varied between0 μM and 30 μM.

A description of the cell viability at different concentrations oftunicamycin without compound or salubrinal is presented in FIG. 23 .

A description of the cell viability in the absence of tunicamycin forcompound ID 1949 and salubrinal is presented in FIG. 24 .

A description of the properties of compound IDs 1901, 1903, 1905, 1906,1912, 1913, 1914, 1918, 1924, and 1946 is presented in FIG. 25 . Theproperties include the total increase in cell viability (AUC) in thepresence of tunicamycin dose-response-induced proteostasis (VialncTot),the total change in cell viability (AUC) caused by compound alone in theabsence of tunicamycin (CmpViaTot), the percent of parent compound lostwhen incubated in phosphate buffer (PlossBuff), the percent of parentcompound converted to salubrinal when incubated in phosphate buffer(SgainBuff), the percent of parent compound lost when incubated in mouseplasma (PlossPlas), the percent of parent compound converted tosalubrinal when incubated in mouse plasma (SgainPlas), the percent ofparent compound lost when incubated in mouse liver microsomes withoutNADPH cofactor (Liver-), and the percent of parent compound lost whenincubated in mouse liver microsomes with NADPH cofactor present(Liver+).

Pharmaceutical Compositions of Dephosphorylation Inhibitors

The dephosphorylation inhibitor may be administered as apharmaceutically acceptable salt, hydrate, or solvate thereof. As usedherein, a “pharmaceutically acceptable salt” or “salt” refers to a saltof one or more compounds. Suitable pharmaceutically acceptable salts ofcompounds include acid addition salts which may, for example, be formedby mixing a solution of the compound with a solution of apharmaceutically acceptable acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoicacid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonicacid, or the like. Where the compounds carry one or more acidicmoieties, pharmaceutically acceptable salts may be formed by treatmentof a solution of the compound with a solution of a pharmaceuticallyacceptable base, such as lithium hydroxide, sodium hydroxide, potassiumhydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodiumcarbonate, potassium carbonate, ammonia, alkylamines, or the like. Asused herein, a “hydrate” refers to a crystal form where a stoichiometricor non-stochiometric amount of water is bound by non-covalentintermolecular forces into the crystal structure. As used herein, a“solvate” refers to a crystal form where a stoichiometric ornon-stoichiometric amount of solvent, or mixture of solvents, isincorporated into the crystal structure. Examples of solvents are water,acetone, ethanol, methanol, propanol, dichloromethane, etc.

In an exemplary embodiment, the compound can be administered in apharmaceutically acceptable carrier. The phrase “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically-acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting a chemical agent. Pharmaceutically acceptablecarriers include pharmaceutically acceptable salts, where the term“pharmaceutically acceptable salts” includes salts of the activecompounds which are prepared with relatively nontoxic acids or bases,depending on the particular substituents found on the compoundsdescribed herein. Examples of pharmaceutically acceptable carriers aresolvents, diluents, dispersion media, suspension aids, surface activeagents, preservatives, solid binders, stabilizers, fillers, bindingagents, lubricants, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Various vehicles and carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof are disclosed in Remington's Pharmaceutical Sciences(A. Osol et al. eds., 15th ed. 1975). Pharmaceutically acceptablecarriers may further comprise minor amounts of auxiliary substances suchas wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the pharmacological agent

Such carriers enable the compounds to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Suitable excipientsare, in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. In one embodiment, mannitol and magnesium stearate are used aspharmaceutically acceptable carriers.

The pharmaceutical compositions may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. In one embodiment, the mode of administrationis oral delivery.

Various solid oral dosage forms can be used for administering compoundsincluding such solid forms as tablets, gelcaps, capsules, caplets,granules, lozenges and bulk powders. The compounds can be administeredalone or combined with various pharmaceutically acceptable carriers,diluents (such as sucrose, mannitol, lactose, starches) and excipientsknown in the art, including but not limited to suspending agents,solubilizers, buffering agents, binders, disintegrants, preservatives,colorants, flavorants, lubricants and the like. Time release capsules,tablets and gels are also advantageous in administering the compounds.In one embodiment, the dephosphorylation inhibitor is administered in ahard gelatin capsule.

Various liquid oral dosage forms can also be used for administeringcompounds, including aqueous and non-aqueous solutions, emulsions,suspensions, syrups, and elixirs. Such dosage forms can also containsuitable inert diluents known in the art such as water and suitableexcipients known in the art such as preservatives, wetting agents,sweeteners, flavorants, as well as agents for emulsifying and/orsuspending the compounds. The compounds may be injected, for example,intravenously, in the form of an isotonic sterile solution. Otherpreparations are also possible. Fingolimod hydrochloride is soluble inwater (>10%) as well as 0.9% saline and aqueous buffers at or below pH2.0. It is very slightly soluble or practically insoluble in aqueousbuffers at or above pH 3.0. A variety of methods are known in the art toimprove the solubility of the pharmacological agent in water and otheraqueous solutions. For example, U.S. Pat. No. 6,008,192 to Al-Razzak etal. teaches a hydrophilic binary system comprising a hydrophilic phaseand a surfactant, or mixture of surfactants, for improving theadministration of lipophilic compounds such as the pharmacologicalagent.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,the pharmacological agent) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile, lyophilized powders for the preparation of sterile injectablesolutions, the method of preparation can include vacuum drying and/orspray-drying to yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts orgelatin.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a pharmacological agent of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the pharmacological agent may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the pharmacological agent to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thepharmacological agent are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a pharmacological agent accordingto the invention is between 5 mg/day to about 2000 mg/day or about 50mg/day to about 1000 mg/day, or in some instances about 100 mg/day to500 mg/day. Preferably, administration of a therapeutically effectiveamount of pharmacological agent, results in a concentration ofpharmacological agent in the bloodstream in the range of 1 nanomolar(nM) to 100 millimolar (mM) concentration. For example, a concentrationrange of about 10 nM to about 10 mM, about, 1 nM to about 1 mM, about 1mM to about 100 micromolar (μM), about 1 μM to about 500 μM, about 1 μMto about 200 μM, preferably about 10 μM to about 50 μM. It is to benoted that the dose may be given in divided doses and dosage values mayvary with the type and severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed composition.

One skilled in the art will appreciate further features and advantagesbased on the above-described embodiments. Accordingly, the methods andcompositions disclosed herein are not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All references, patents, patent applications and otherpublications cited herein are expressly incorporated herein in theirentirety.

While the applicant's teachings are described in conjunction withvarious embodiments, it is not intended that the applicant's teachingsbe limited to such embodiments. On the contrary, the applicant'steachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

The invention claimed is:
 1. A method comprising administering acompound of Formula (II) to a subject having a neurological disorder,wherein the compound of Formula (II) has the structure:

wherein R¹ is H, R² is H, R³ is H, R⁴ is H or null, R⁵ is —SR⁶, R⁶ isCH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆ cycloheteroalkyl, amino acid, aminoacid derivative, C₁₋₂₀ alkyl, or C₁₋₂₀ heteroalkyl, A-B is C═CH, and D-Eis CH—N or C═N; and wherein the neurological disorder is amyotrophiclateral sclerosis (ALS), multiple sclerosis (MS), Alzheimer's disease,Parkinson's disease, Huntington's disease, prion-associated disease,spinocerebellar ataxia, or spinal muscular atrophy.
 2. The method ofclaim 1, wherein the compound is selected from the group consisting of:


3. The method of claim 2, wherein the compound is a hydrochloride saltor a phosphate salt thereof.
 4. The method of claim 1, wherein R¹ is H,R² is H, R³ is H, R⁴ is H, CH₂(C₃₋₆ cycloheteroalkyl), C₃₋₆cycloheteroalkyl, amino acid, amino acid derivative, C₁₋₂₀ alkyl, orC₁₋₂₀ heteroalkyl, R⁵ is ═S, A-B is C═CH, and D-E is C—N.
 5. The methodof claim 1, wherein the compound is a hydrochloride salt or a phosphatesalt thereof.
 6. The method of claim 1 further comprising inhibitingendoplasmic reticulum stress-mediated apoptosis.
 7. The method of claim6, wherein the endoplasmic reticulum stress-mediated apoptosis isinduced by at least one protein glycosylation inhibitor or at least onphosphatase enzyme.
 8. The method of claim 7, wherein the proteinglycosylation inhibitor is tunicamycin.
 9. The method of claim 7,wherein the phosphatase enzyme is eukaryotic translation initiationfactor 2 alpha (eIF2α).
 10. The method of claim 1, wherein the compoundis formulated with a pharmaceutically acceptable diluent, adjuvant, orcarrier.
 11. The method of claim 1, wherein the compound is formulatedfor oral administration.
 12. The method of claim 1, wherein the compoundis formulated as a single daily dose.
 13. The method of claim 1, whereinthe compound is formulated in a dosage between about 5 mg and about 2000mg.